Endo S2 (200U/μL)

Abstract: Monoclonal antibodies (mAb) produced in 1,4-mannosyl-glycoprotein 4-N-acetylglucosaminyltransferase (MGAT3) overexpressing cell lines have superior in vitro and in vivo activities. The N-glycan of the Fc-region of these mAbs have increased levels of bisecting N-acetylglucosamine (GlcNAc) and reduced core-fucosylation. Although a reduction in core-fucosylation will improve FcγRIIIa binding and antibody-dependent cellular cytotoxicity (ADCC) activity, the influence of bisecting GlcNAc on these activities has been difficult to probe. Here, we describe the preparation of a unique series of homogeneous glycoforms of trastuzumab (Herceptin) with and without core-fucose and with and without bisecting GlcNAc and examine binding to a comprehensive panel of Fcγ receptors. The glycoforms of trastuzumab were prepared by treatment with wild-type Endo-S2 to cleave the chitobiose core of the N-glycan to leave GlcNAc-Fuc that was exposed to an α-fucosidase to provide trastuzumab-GlcNAc. Glycan oxazolines with and without bisecting GlcNAc were prepared by enzymatic remodeling of a sialoglycopeptide isolated from egg yolk powder, which were employed in transglycosylations with trastuzumab-GlcNAc and trastuzumab-GlcNAc-Fuc catalyzed by Endo-S2 D184M resulting in well-defined glycoforms. As expected, core-fucosylation had a major effect on FcγRIIIa binding, which was not influenced by the presence of bisecting GlcNAc. It was found that an A2-glycan (GlcNAc2Man3GlcNAc2) modified by bisecting GlcNAc cannot be core-fucosylated by FUT8. Thus, bisecting GlcNAc has only an indirect influence on FcγRIIIa binding and subsequent ADCC activity by inhibiting core-fucosylation. The results described here provide an understanding of the properties of therapeutic monoclonal antibodies.

Abstract: Sulfated fucan, an important marine polysaccharide frequently presented in echinoderms and brown algae, has gained growing attention owing to its various biological activities. Fucanases are essential tools for degrading sulfated fucan to produce corresponding oligosaccharides. In this context, an endo-1,3-fucanase (Fun187Al) belonging to the GH187 family was successfully expressed in Escherichia coli. Fun187Al showed the highest activity at 30-40 °C and pH 7.5. It hydrolyzed sulfated fucan in a random endo-acting pattern, and displayed a substrate specificity different from the endo-1,3-fucanases of other glycoside hydrolase family. Analyses of ultra-performance liquid chromatography coupled with high-resolution mass spectrometry revealed that tetrasaccharide with two sulfate groups (Fuc4S2), Fuc4S3, and Fuc4S4 were respectively the major components in the end products of Fun187Al against sulfated fucans from Acaudina molpadioides, Thelonota ananas, and Holothuria tubulosa. The capability of Fun187Al to produce oligosaccharides with different degrees of polymerization and sulfation patterns demonstrated that it could be regarded as a favorable tool for establishing the structure-activity relationships of sulfated fucan and its oligosaccharides.Copyright © 2025 Elsevier B.V. All rights reserved.

Abstract: This study evaluated the extent to which obturation materials bypass fractured endodontic instruments positioned in the middle and apical thirds of severely curved simulated root canals using different obturation techniques. Sixty resin blocks with simulated root canals were used, each with a 50° curvature, a 6.5 mm radius of curvature, and a length of 16.5 mm, prepared to an ISO #15 diameter and taper. Canals were shaped using ProTaper Universal files (Dentsply Maillefer) attached to an X-smart Plus endo motor (Dentsply), set at 3.5 Ncm torque and 250 rpm, up to size S2 at working length. To simulate fractures, F2 and F3 files were weakened 3 mm from the tip, then twisted to break in the apical and middle sections of the canal, respectively. All samples were sealed with GuttaFlow 2 and divided into three groups (n = 20/group) according to obturation technique: A) single cone, B) lateral condensation with a rotary spreader, and C) softcore obturators. Each group was then divided into two subgroups (n = 10) based on the instrument fracture location (1 = apical, 2 = middle). The linear intrusion of obturation materials through the fractured instruments was measured using ImageJ software and analyzed statistically with ANOVA, Tukey tests, and independent t-tests, with significance set at p<0.05. Material bypass in group B1 (3.27 ± 0.63 mm) was significantly greater than in group A1 (2.39 ± 0.44 mm) and group C1 (2.91 ± 0.77 mm). In group C2, bypass (5.76 ± 0.64 mm) was significantly higher than in group A2 (3.82 ± 0.2 mm) and group B2 (2.27 ± 0.96 mm). Additionally, bypass in group A2 was greater than in group B2, and group B1 had more bypass than B2, while group C2 exceeded C1. The lateral condensation technique with a rotary spreader and softcore obturators increased the bypass of obturation materials through fractured instruments in simulated curved canals. These techniques may thus enhance material flow in endodontic procedures involving instrument fractures.Copyright: © 2025 Lateef et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract: The intracanal antibacterial effectiveness of a bioceramic medication was compared with calcium hydroxide pastes in different vehicles.Extracted mandibular incisors with a single long oval canal were selected and distributed into 5 groups based on anatomically paired microcomputed tomographic analyses. The root canals were prepared up to an instrument size 35/04 and contaminated for 30 days with a mixed bacterial culture from subgingival biofilm added with Enterococcus faecalis. The canals were medicated with Bio-C Temp (Angelus Indústria de Produtos Odontológicos, Londrina, PR, Brazil) or a calcium hydroxide paste in glycerin, camphorated paramonochlorophenol and glycerin (CHPG), or 2% chlorhexidine (CHCX). Control specimens were filled with sterile saline. After 7 days, the medication was removed by using rotary instruments and saline irrigation. A supplementary approach was conducted, which consisted of 2.5% sodium hypochlorite irrigation and agitation with XP-endo Finisher. Bacteriological samples were taken before application (S1) and after removal (S2) of the intracanal medication, and after the supplementary approach (S3). Bacterial DNA extracted from the samples was quantified using real-time polymerase chain reaction.Bacterial counts were significantly reduced from S1 to S2 in all groups, except for the control group. As for S1-to-S3 changes, all groups (control included) showed significant bacterial reduction. S2-to-S3 changes were only significant in the CHPG and CHCX groups (P < .05). Intergroup comparisons showed no significant differences in S2 (P > .05), with all medications significantly better than the control (P < .05). In S3, only the CHPG and CHCX groups showed further significant bacterial reductions. Categorical data analysis showed no intergroup differences (P > .05).All tested medications significantly reduced the intracanal bacterial counts, with no significant differences between them. Final sodium hypochlorite irrigation and agitation using XP-endo Finisher further enhanced disinfection in the CHCX and CHPG groups.Copyright © 2024 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.